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Flow cytometric determination of PMCA‐mediated Ca2+‐extrusion in individual red blood cells
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AbstractBackground:Differences among red blood cells in the activity of the plasma membrane Ca2+‐ATPase (PMCA) can impact cell signaling and survival. However, no method has been reported that measures this activity directly in individual cells.Methods:We have designed a novel assay for PMCA activity that uses the fluorescent Ca2+‐reporter Fluo4 and flow cytometric analysis. The method recognizes the extrusion of Ca2+ from the cell after a short Ca2+‐loading pulse, which avoids the problem of ATP depletion and ascertains activity at Vmax capacity.Results:Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca2+‐pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity. Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA Vmax.Conclusion:We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells. © 2007 International Society for Analytical Cytology
Title: Flow cytometric determination of PMCA‐mediated Ca2+‐extrusion in individual red blood cells
Description:
AbstractBackground:Differences among red blood cells in the activity of the plasma membrane Ca2+‐ATPase (PMCA) can impact cell signaling and survival.
However, no method has been reported that measures this activity directly in individual cells.
Methods:We have designed a novel assay for PMCA activity that uses the fluorescent Ca2+‐reporter Fluo4 and flow cytometric analysis.
The method recognizes the extrusion of Ca2+ from the cell after a short Ca2+‐loading pulse, which avoids the problem of ATP depletion and ascertains activity at Vmax capacity.
Results:Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca2+‐pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity.
Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA Vmax.
Conclusion:We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells.
© 2007 International Society for Analytical Cytology.
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